Functional analysis of the cellular RNA directed RNA polymerase (RdRP) in higher plants [Elektronische Ressource] / vorgelegt von Fareha Razvi

Lukow

Sujets

• Biologie

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2003
• Nombre de lectures: 16
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Functional analysis of the cellular RNA-directed RNA
polymerase (RdRP) in higher plants










Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der Rheinisch-
Westfälischen Technischen Hochschule Aachen zur Erlangung des akademischen Grades
einer Doktorin der Naturwissenschaften genehmigte Dissertation




vorgelegt von




Master of Science

Fareha Razvi

aus New Delhi (India)




Berichter: Universitätsprofessor Dr. rer. nat. Rainer Fischer
Universitätsprofessor Dr. rer. nat. Wolfgang Nellen


thTag der mündlichen Prüfung: 17 June 2003
Diese Dissertation ist auf den Internetseiten
der Hochschulbibliothek online verfügbar
















Dedicated to
my paternal and maternal
grandparents

“Everything soft and tender reminds me of you”

Although I never saw my grandparents, I feel their presence would have
made a difference to my life...
Acknowledgements

I wish to place on record my deep gratitude to Prof. Dr. Rainer Fischer for giving me an
opportunity to join his group and commence research in the area that captured my interest and
imagination at first glance. His unbridled support and encouragement have contributed a lot in
sustaining my efforts.

I am indebted to Dr. Michael Wassenegger for his hospitality at the inception of my stay in
Munich. I thank him very much for his discussions, support, editorial judgement and insightful
contributions to lend finesse to every aspect of my draft.

I thank Prof. Dr. Wolfgang Nellen for reviewing the thesis and agreeing to be the co-examiner.

I wish to extend my thanks to Dr. Arno Pütz and Dr. Ulrike Vogt, for delightful discussions and for
their in-time help outside and inside of the laboratory. I thank Dr. J. Nähring, Mr. Lars Giesen, Dr.
Thomas Lukow, Dr. J. Muth and his group, for providing the DNA sequencing facility.

I thank Sabine, Slyvia, Katja and Klaus for their amicable technical help and many thanks to Frau
Anna Maria Bauer and Frau Lydia Biock-Oswald for maintenance and propagation of the
transgenic plants in green house, Munich.

I would like to express my deep appreciation of the help provided by Dr. Ralf Tatzel and Frau G.
Weingaertner from Max Planck Institute für Biochemie, Munich.

I am extremely grateful to my Lecturers and Professors from Delhi University for their conceptual
teachings, discussion and valuable suggestions that helped me acquire scientific temperament. A
special mention of Prof. G. P. Talwar, who gave me an opportunity to get familiar with the
fundamentals of research.

I owe special thanks to Dr. Raj Bhatnagar of International Centre for Genetic Engineering and
Biotechnology, Delhi, whose continued support, encouragement and friendly discussions
contributed to development of interest in research.

Jaikrit, Geetanjali and Mukta, for keeping my spirits up and my hamstrings in working order. I
wish to thank specially for their cheerfulness and forbearance during the typing of this thesis.
Special thanks to my friends Dr. Fuentes-Prior, Thomas, Dr. Pandurangan and Dr. Archana for
critical review of my thesis. I thank all my friends in India for their encouraging words.

Finally but immensely, I owe major debts to “The backbone of my life”-my loving parents and
caring brothers, whose immeasurable support and eminent understanding has helped me through
thick and thin. Pumping me with enthusiasm all through the years. With their inexplicable
contributions only, I have reached this stage. I thank them for being beside me. Further, I wish to
thank Farina for sharing pleasant moments of life, Tabinda and Amina my nieces, for being mine.
I
TABLE OF CONTENTS



I Introduction 1
I.1 Recognition of RdRPs 3
I.2 The principle of PTGS 4
I.2.1 PTGS and its counterpart
I.2.2 The mechanism of PTGS/RNAi 6
I.2.3 Silencing a powerful tool for functional genomics 8
I.3 Aim of the thesis 10

II Materials and Methods 14
II.1 Materials
II.1.1 Chemicals and consumables 14
II.1.2 Enzymes and reaction Kits 14
II.1.3 Host strains 15
II.1.4 Plants
II.1.5 Vectors 15
II.1.6 Oligonucleotides 16
II.1.7 Buffers, media and solutions
II.1.8 Matrices and membranes 17
II.1.9 Equipments 17
II.2 Methods
II.2.1 Standard molecular cloning 18
II.2.1.1 DNA modifications
II.2.1.1.1 Plasmid DNA restriction 18
II.2.1.1.2 T4 DNA Polymerase fill-in
II.2.1.2 Agarose gel electrophoresis 18
II.2.1.3 Gel extraction 19
II.2.1.4 Ligation
II.2.1.5 Preparation and transformation of competent Escherichia coli cells 19
II.2.1.6 Identification and analysis of recombinant plasmids 21
II
II.2.1.6.1 Pooled polymerase chain reaction (pooled PCR) of E. coli
transformants 21
II.2.1.6.2 Plasmid DNA mini-preparation 22
II.2.1.6.3 Mini-preparation of Agrobacterium DNA
II.2.1.7 Polymerase Chain Reaction -TaKaRa Ex Taq 22
II.2.1.7.1 Standard PCR Reaction Mix
II.2.1.7.2 Standard PCR Conditions 23
II.2.1.7.3 5’/3’ Rapid amplification of cDNA ends (RACE)
II.2.1.7.4 cDNA synthesis 23
II.2.1.7.5 Re-amplification of PCR products
II.2.1.8 Cryopreservation of the bacterial cells 25
II.2.2 Generation and characterisation of transgenic tobacco plants 25
II.2.2.1 Conjugation 25
II.2.2.2 Re-transformation into INV α F´
II.2.2.3 Agrobacterium-mediated “leaf disc transformation” of tobacco plant 26
II.2.2.4 Self-pollination, genetic crosses and sterilisation of seeds 26
II.2.3 Growth of transgenic plants 28
II.2.4 Characterisation of transgenic plants 29
II.2.4.1 Extraction of genomic DNA from plants
II.2.4.2 Southern blot analysis 30
II.2.4.3 RNA isolation from plants 31
II.2.4.4 Northern blot analysis
II.2.4.5 Radiolabelled DNA probes 34
II.2.4.6 Inoculation of plants with the in vitro transcripts 34

III Results 35
III.1 Amino acid alignment of the RNA-directed RNA polymerase (RdRP) homologues
within and across plant species 35
III.1.1 Data base search and alignment
III.1.2 A tomato EST comprising sequence homology with the Arabidopsis
2 thaliana RdRP 36
2III.1.3 Isolation of a RdRP -specific cDNA fragment from tomat 37

III
2III.1.4 Isolation of a 3’end-specific fragment of the RdRP gene from tomato and
tobacco 38
2III.1.5 Isolation of the RdRP 5’end from tomato 40
1 2III.2 Designing of RdRP and RdRP transgene constructs for stable plant
transformation 44
III.2.1 Designing of repeat constructs for endogenous RdRP suppression 45
III.2.1.1 Isolation and cloning of RdRP-specific 3’end fragments 45
2III.2.1.1.1 Isolation and cloning of RdRP - specific 3’end fragments
1III.2.1.1.2 Isolation and cloning of RdRP -specific 3’end fragments 51
III.2.2 Designing of the sense and antisense constructs to suppress expression
1 2of RdRP and RdRP 57
III.2.2.1 Isolation and cloning of RdRP-specific 3’end fragments into the
pPCV702SM vector 57
2III.2.2.1.1 Cloning of RdRP -specific 3’end fragments
1III.2.2.1.2 ents 59
2III.2.3 Designing of the sense constructs to over-express RdRP 62
2III.2.3.1 Cloning of the full-length RdRP cDNA into the pPCV702SM vector 62
2III.2.3.1.1 Assembly of the tomato full-length RdRP cDNA 62
2III.2.3.1.2 Introduction of the full-length RdRP cDNA into the pPCV702SM
vector 63
III.3 Generation and characterisation of transgenic Nicotiana tabacum plants 65
III.3.1 Characterisation of transgenic plants 67
III.3.1.1 Southern analysis of primary transformants displaying no obvious
phenotypic alterations 67
2III.3.1.1.1 Southern analysis of plant lines transformed with the RdRP repeat
T-DNA constructs 67
1III.3.1.1.2 ed with the RdRP direct
repeat T-DNA construct 76
2III.3.1.1.3 ed with the RdRP 3’end-
containing T-DNA constructs